A Se-containing hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii. The molecular weight of the enzyme was estimated as 340,000. The enzyme tends to aggregate and occurs also as a larger protein species. Both molecular species reduce the 8-hydroxy-5-deazaflavin cofactor with molecular hydrogen. Se is present exclusively in Mr = 42,000 subunit. A value of 3.8 g atoms of Se/mol of enzyme (Mr = 340,000) was determined. The chemical form of selenium in the enzyme was shown to be selenocysteine. This is the first hydrogenase that has been shown to be a selenoenzyme.